DE102022125712B3 - Method for estimating the germination properties of plant seeds and test kits - Google Patents

Abstract

The invention relates to a method for estimating the germination properties of plant seeds, comprising the method steps: a) providing a plurality of separate plant seed portions, each comprising at least one plant seed, b) producing or providing a test composition comprising: i) water, ii) a two-stage redox indicator, and iii) a fermenting microorganism, c) contacting the plant seed portions with a test volume of test composition to obtain a large number of separate test systems and incubating the test systems, d) measuring the test compositions of the incubated test systems using an optical measuring method to determine the optical absorption properties of the respective test compositions for electromagnetic radiation at least a first wavelength λ1 and a second wavelength λ2 to obtain a plurality of absorption data sets assigned to the respective test systems, the absorption data sets comprising information about the absorption properties of the respective test compositions at the first wavelength λ1 and the second wavelength λ2, where λ1 and λ2 are around 10 nm or more, e) evaluating the absorption data sets assigned to the test systems to estimate the germination properties of the plant seeds in the respective plant seed portions with an estimation module based on machine learning.

Description

The invention relates to a method for estimating the germination properties of plant seeds, a test composition for use in such a method, a kit for producing a corresponding test composition and a computer program product for carrying out steps of the corresponding method. An electronic data processing device for use in such a method is also disclosed.

Providing a reliable supply of sufficient food to the world's growing population is one of the most pressing challenges facing humanity. Against this background, past and future innovations in the field of agriculture and agricultural technology are of particular importance. For many of the most important agricultural products, access to high-performance seeds that result in usable plants reliably and with high yields is essential in order to achieve maximum yield on the available land.

Against this background, seeds represent an important economic commodity in the agricultural sector that is subject to high demands. These requirements relate in particular to the ability of the seeds to have sufficient germination and growth potential after sowing. The quality assessment of seeds and the guarantee of consistent seed qualities play such an important role that the “Association of Official Seed Analysts” (AOSA) and the “International Seed Testing Association” (ISTA) have been international associations since 1908 and 1924, respectively who are committed to seed quality as well as their testing and certification.

The tests defined by ISTA to determine the germination properties of seeds and the defined quality criteria represent the undisputed industry standard in most countries of the European Union and are continuously updated by ISTA. Accordingly, the present invention focuses primarily on the ISTA methods and criteria, although in many cases these differ only slightly from the AOSA criteria.

In a shortened summary, the classic methods for determining germination properties are based on the removal of a representative seed fraction from a batch of seeds to be evaluated, with the test seeds thus obtained being exposed to conditions under which the seeds can germinate, the presence of germination and the extent of the driving force being determined are determined for the individual seeds of the test group and the germination properties characteristic of the seed batch are derived through stochastic evaluation.

Even if the tests specified by the ISTA have undisputedly the greatest relevance to the industry to date, determining the germination properties through the actual germination of the seeds is in many cases perceived as disadvantageous given the time and effort required. Against this background, there is continued interest in providing methods with which the germination properties of plant seeds can be reliably and quickly estimated, aiming for a satisfactory correlation with the germination properties to be obtained according to ISTA.

A germination test for plant seeds known from the prior art is, for example, in DE 10 2020 200 567 A1 disclosed. This germination test is based on contacting a number of plant seeds in separate vessels with a predetermined volume of a test solution comprising resazurin, ie a two-stage redox indicator, and yeast, ie a fermenting microorganism. The plant seeds are contacted with the test solution and incubated. In general, it is assumed that the germination ability of plant seeds decreases as the damage to the seed coat increases. This damage to the seed coat leads to a leakage of glucose and other organic substances as well as inorganic substances from the inside of the seed into the surrounding test solution during incubation. These substances can be at least partially converted by the yeast, which results in a change in the two-stage redox indicator resazurin to resorufin or dihydroresorufin. This implementation leads to a color change. The DE 10 2020 200 567 A1 exploits this by measuring the absorbance of the test solution at 570 nm to correlate the normalized absorbance values with the actual germination properties obtained in germination tests, which are subsequently determined. Linear regression can be used to estimate functions, which in turn can be used to estimate the germination properties for similar plant seeds from absorption values. The procedure of DE 10 2020 200 567 A1 According to the inventors' assessment, it basically represents a wide variety speaking germination test is available, which can be carried out significantly faster than the methods based on actual sowing. This process, which enables at least a rough estimate of the germination properties for a wide range of plant seeds, also brings advantages in terms of time and cost efficiency, is non-destructive and in most cases causes only minor environmental impact.

Despite the advantages described above of the method known from the prior art, many experts in the art - including the inventors of the present invention - feel that it needs improvement in many respects. In particular, the prediction quality that can be achieved with the prior art method is regularly perceived as insufficient, especially because very large data sets are usually required to obtain reliable correlations between the absorption at 570 nm and the germination capacity, despite sometimes complex normalization processes a large spread around the regressions obtained is observed. The inventors assume that it is this inadequacy in the central functionality, i.e. the assessment of germination properties, that is why the inherently advantageous method for analyzing the germination ability of plant seeds is still in use to this day, despite the advantages described above has not yet been fully established in the industry.

The primary object of the present invention was to eliminate or at least mitigate the disadvantages of the prior art described above.

In particular, it was the object of the present invention to provide a method for estimating the germination properties of plant seeds, with which the germination properties of plant seeds can be estimated particularly reliably and precisely.

It was an object of the present invention that the method to be specified should be able to be carried out as a non-invasive method which does not destroy the plant seeds examined and which should desirably be as unproblematic as possible with regard to environmental and health aspects.

In addition, it was an object of the present invention that the method to be specified should enable the estimation of the germination properties and the evaluation of the seed quality significantly faster than the germination tests known from the prior art, so that the method to be specified should have a higher time and cost efficiency.

It was a supplementary object of the present invention that the method to be specified should be able to be operated efficiently in an at least partially decentralized configuration, so that the assessment of the germination properties of plant seeds can also be carried out outside of research institutions and with the lowest possible need for equipment .

It was a further object of the present invention that the method to be specified should be applicable to a wide range of possible plant seeds and should enable a reliable assessment of the germination properties regardless of the plant seeds used.

It was an object of the present invention that the method to be specified should be able to reliably estimate the germination properties, in particular the germination capacity and the driving force.

It was a further object of the present invention that the method to be specified should be easily automated and suitable for high throughput rates. In addition, the method to be specified should desirably be able to be interpreted in such a way that it enables particularly easy handling and should place comparatively low requirements with regard to the education and training of the workers used in the method.

It was a further object of the present invention to provide a particularly advantageous test liquid for use in the method to be specified and a kit for producing plant seed-specific test compositions.

In addition, it was a further object of the present invention to provide a computer program product with which essential method steps of the method to be specified can be carried out on an electrical data processing device.

It was a secondary object of the present invention to provide an electronic data processing device which is suitable for use in the method to be specified.

The inventors of the present invention have now recognized that the tasks described above can surprisingly be achieved if, starting from a germination test known from the prior art, which relies on the use of resazurin and yeast and the photometric evaluation of the color change of resazurin at 570 nm based, rather the absorption properties are detected at two sufficiently different wavelengths and the absorption data sets obtained in this way are evaluated using machine learning techniques, as defined in the claims.

Surprisingly, this modification of the method known from the prior art, i.e. H. the detection of at least two sufficiently separated wavelengths and the use of machine learning methods, advantageous methods are obtained which can estimate the germination properties of plant seeds with significantly improved forecasting accuracy, the resulting method being fast, reliable, inexpensive, harmless and seed-preserving and can be used particularly in decentralized configurations with comparatively low expenditure on equipment and low requirements for the level of training of the workforce used. Compared to the germination test known from the prior art, a significant and consistent improvement in the prediction of the most important germination properties is achieved.

The above-mentioned objects are thus achieved by the subject matter of the invention, as defined in the claims. Preferred embodiments according to the invention result from the subclaims and the following statements.

Such embodiments, which are referred to below as preferred, are combined in particularly preferred embodiments with features of other embodiments designated as preferred. Combinations of two or more of the embodiments described below as particularly preferred are therefore very particularly preferred. Also preferred are embodiments in which a feature of one embodiment that is designated as preferred to some extent is combined with one or more further features of other embodiments that are designated as preferred to some extent. Features of preferred test compositions, kits, electronic data processing devices and computer program products arise from the features of preferred methods.

Insofar as both specific amounts or proportions of this element and preferred embodiments of the element are disclosed below for an element, for example for the two-stage redox indicator or the fermenting microorganism, in particular the specific amounts or proportions of the preferably designed elements are also disclosed. In addition, it is disclosed that in the corresponding specific total quantities or total proportions of the elements, at least some of the elements can be preferably designed and in particular also that preferably designed elements within the specific total quantities or total proportions can in turn be present in the specific quantities or proportions.

• a) Bereitstellen einer Vielzahl von separaten Pflanzensamenportionen umfassend jeweils zumindest einen Pflanzensamen,
• b) Herstellen oder Bereitstellen einer Testzusammensetzung, umfassend:
  • i) Wasser,
  • ii) einen zweistufigen Redox-Indikator, und
  • iii) einen fermentierenden Mikroorganismus,
  • c) Kontaktieren der Pflanzensamenportionen mit jeweils einem Testvolumen an Testzusammensetzung zum Erhalt einer Vielzahl von separaten Testsystemen und Inkubieren der Testsysteme,
  • d) Vermessen der Testzusammensetzungen der inkubierten Testsysteme mit einem optischen Messverfahren zur Bestimmung der optischen Absorptionseigenschaften der jeweiligen Testzusammensetzungen für elektromagnetische Strahlung zumindest einer ersten Wellenlänge λ₁ und einer zweiten Wellenlänge λ₂ zum Erhalt einer Vielzahl an den jeweiligen Testsystemen zugeordneten Absorptionsdatensätzen,
  wobei die Absorptionsdatensätze Informationen über die Absorptionseigenschaften der jeweiligen Testzusammensetzungen bei der ersten Wellenlänge λ₁ und der zweiten Wellenlänge λ₂ umfassen, wobei sich λ₁ und λ₂ um 10 nm oder mehr unterscheiden,
• e) Auswerten der den Testsystemen zugeordneten Absorptionsdatensätze zur Abschätzung der Keimungseigenschaften der Pflanzensamen in den jeweiligen Pflanzensamenportionen mit einer elektronischen Datenverarbeitungsvorrichtung, wobei die elektronische Datenverarbeitungsvorrichtung eine Speichereinheit umfasst, wobei auf der Speichereinheit ein auf maschinellem Lernen basierendes Abschätzungsmodul gespeichert ist, wobei die elektronische Datenverarbeitungsvorrichtung dazu eingerichtet ist, die für die Pflanzensamenportionen erhaltenen Absorptionsdatensätze als Eingabe in das Abschätzungsmodul zu geben und die Keimungseigenschaften der Pflanzensamen in den Pflanzensamenportionen mit dem Abschätzungsmodul abzuschätzen, wobei das Abschätzungsmodul dazu trainiert ist, aus den Absorptionsdatensätzen die Keimungseigenschaften der Pflanzensamen in den Pflanzensamenportionen abzuschätzen, wobei das Training mittels überwachtem Lernen mit einem Satz von Trainingsdaten erfolgt, welcher eine Vielzahl von Trainingsabsorptionsdatensätzen von Trainingspflanzensamenportionen von Pflanzensamen mit bekannten Keimungseigenschaften umfasst.

The invention relates to a method for estimating the germination properties of plant seeds, comprising the method steps:

• a) providing a plurality of separate plant seed portions, each comprising at least one plant seed,
• b) producing or providing a test composition comprising:
  • i) water,
  • ii) a two-stage redox indicator, and
  • iii) a fermenting microorganism,
  • c) contacting the plant seed portions each with a test volume of test composition to obtain a large number of separate test systems and incubating the test systems,
  • d) measuring the test compositions of the incubated test systems using an optical measurement method for determining the optical absorption properties of the respective test compositions for electromagnetic radiation of at least a first wavelength λ ₁ and a second wavelength λ ₂ in order to obtain a large number of absorption data sets assigned to the respective test systems,
  wherein the absorption data sets include information about the absorption properties of the respective test compositions at the first wavelength λ ₁ and the second wavelength λ ₂ , where λ ₁ and λ ₂ differ by 10 nm or more,
• e) evaluating the absorption data sets assigned to the test systems for estimating the germination properties of the plant seeds in the respective plant seed portions with an electronic data processing device, the electronic data processing device comprising a storage unit, an estimation module based on machine learning being stored on the storage unit, the electronic data processing device being set up for this purpose is to enter the absorption data sets obtained for the plant seed portions as input into the estimation module and to estimate the germination properties of the plant seeds in the plant seed portions with the estimation module, the estimation module being trained to estimate the germination properties of the plant seeds in the plant seed portions from the absorption data sets, the training by means of supervised learning with a set of training data, which includes a large number of training absorption data sets of training plant seed portions of plant seeds with known germination properties.

The method according to the invention is used to estimate the germination properties of plant seeds. Linguistically, this formulation is in contrast to that in the DE 10 2020 200 567 A1 However, the wording used for a “germinability test” seems more appropriate to the inventors. This is because, strictly speaking, the germination properties of the plant seeds are not tested or analyzed in the method according to the invention, as would be the case, for example, in ISTA tests. Rather, the method according to the invention relies on predicting the germination properties of plant seeds that have not been germinated by correlating them with absorption values measured on subsequently germinated, similar plant seeds, so that the actual test of the germination properties can be avoided. The expression “germination properties” expresses, in accordance with the expert understanding, that analogous to the various criteria developed by ISTA, in addition to germination, other parameters can also be assigned to the germination properties, namely, for example, the driving force, whereby these properties are also advantageous Way can be determined using the method according to the invention. An example is therefore a method according to the invention, wherein the estimated germination properties each indicate an estimate of the probability that the plant seeds of the plant seed portion will germinate physiologically, preferably the probability that the plant seeds of the plant seed portion will germinate physiologically within a predetermined observation period after sowing, whereby the presence physiological germination is particularly preferably checked every 24 hours. In accordance with professional understanding, “physiological germination” can be understood as a breakthrough of a root from a plant seed with a length of at least 2 mm, although a so-called “white root tip” is sometimes also referred to. In practice, however, the exact criteria will be based on the current ISTA criteria. Additionally or alternatively, an example is also a method according to the invention, wherein the estimated germination properties each include an estimate of the probability that the plant seeds of the plant seed portion will form a normal seedling in the sense of the ISTA criteria. A normal seedling in the sense of the current ISTA criteria is when a seed forms a healthy, complete seedling that can be expected to develop into a complete plant.

The person skilled in the art understands that the method according to the invention can be used efficiently to determine the germination properties of a large number of plant seeds. In practice, analogous to the method developed by ISTA, the most relevant application will be in which the germination properties of a large batch of seeds are determined by analyzing a representative subset of plant seeds.

In the method according to the invention, a large number of plant seeds are first provided in method step a), which can, for example, be a corresponding representative subset of a larger batch of seeds, these plant seeds being divided into separate plant seed portions. To avoid the influence of any contamination on the surface of the plant seeds, it is usually advisable to at least roughly clean the plant seeds before the process, for example by washing them with distilled water.

As further specified below, in the opinion of the inventors, the method according to the invention will in most cases be used to identify an overall evaluation parameter for the entirety of the plant seeds, i.e. H. to derive a parameter of the entirety (“bulk property”) or an average germination property prediction for the seed batch. Against this background, it is fundamentally possible to use two or more plant seeds together as plant seed portions, so that, for example, 100 plant seed portions can be used, each with two plant seeds per plant seed portion. According to the inventors' assessment, however, with a view to the achievable resolution of the germination properties, it is preferred to use separate plant seeds as the plant seed portion, since this prevents differences in the germination properties of the plant seeds from being lost by averaging, as would be the case, for example, if a plant seed with a particularly advantageous germination capacity would be measured together with a plant seed with a low germination capacity in a common plant seed portion. Preferred for essentially all embodiments is, in accordance with the expert's understanding of efficient experimental planning, a method according to the invention, wherein the plant seed portions comprise the same number of plant seeds. Particularly preferred for essentially all embodiments is a method according to the invention, wherein the plant seed portions consist of one or two, preferably one plant seed, and/or wherein each test system comprises exactly one plant seed.

In method step b), a test composition is produced or provided, the latter can be done, for example, by purchasing a finished test composition from a supplier. However, according to the inventors' assessment, in the majority of cases the test composition will be subject to certain signs of aging, so that in view of the achievable estimation quality it is particularly preferred to produce the test composition using the method according to the invention.

This can be done, for example, by mixing the two-stage redox indicator and the fermenting microorganism with water immediately before carrying out the further process steps in order to produce the test composition. This procedure has the great advantage that unwanted changes in part of the redox indicator, which could occur as an aging phenomenon in the test composition, can be avoided. Accordingly, preference is given to a method according to the invention, wherein the test composition is prepared in method step b), the test composition being prepared preferably 2 hours or less, particularly preferably 1 hour or less, very particularly preferably 0.5 hour or less, particularly preferably immediately after contacting the plant seed portions.

• - Süßgräsern (Poaceae), beispielsweise Alopecurus, Avena, Hordeum, Lolium, Poa, Secale, Triticum, Triticale, Zea;
• - Amaryllisgewächsen (Amaryllidaceae), beispielsweise Allium;
• - Spargelgewächsen (Asparagaceae), beispielsweise Asparagus;
• - Apiaceae, beispielsweise Apium, Daucus, Petroselinum;
• - Asteraceae, beispielsweise Helianthus;
• - Brassicaceae, beispielsweise Brassica, Lepidium, Raphanus, Sinapis;
• - Caprifoliaceae, beispielsweise Valerianella;
• - Chenopodiaceae, beispielsweise Beta;
• - Cucurbitaceae, beispielsweise Cucumis;
• - Fabaceae, beispielsweise Glycine. Lens, Pisum, Trifolium, Vicia, Lupinus;
• - Lamiaceae, beispielsweise Ajuga, Lavendula;
• - Ranunculaceae, beispielsweise Delphinium;
• - Rosaceae, beispielsweise Fragaria, Filipendula;
• - Solanaceae, beispielsweise Lycopersicum, Solanum.

The method according to the invention is advantageously suitable for use with a wide range of plant seeds. In fact, based on the test data available, the inventors have no reason to assume that the method according to the invention could not be applicable to certain types of plant seeds. The inventors have demonstrated its applicability for a wide range of different types of plant seeds through extensive testing. In this respect, preference is therefore given to a method according to the invention, wherein the plant seeds are seeds of plants which are selected from the group consisting of plant seeds of gymnosperms (Gymnospermae) and angiosperms (Angiospermae), preferably selected from the group consisting of plant seeds of gymnosperms (Gymnospermae). , monocotyledonous angiosperms (Monocotyledonae) and dicotyledonous angiosperms (Dicotyledonae), particularly preferably selected from the group consisting of plant seeds from:

• - Poaceae, for example Alopecurus, Avena, Hordeum, Lolium, Poa, Secale, Triticum, Triticale, Zea;
• - Amaryllis family (Amaryllidaceae), for example Allium;
• - Asparagus family (Asparagaceae), for example Asparagus;
• - Apiaceae, for example Apium, Daucus, Petroselinum;
• - Asteraceae, for example Helianthus;
• - Brassicaceae, for example Brassica, Lepidium, Raphanus, Sinapis;
• - Caprifoliaceae, for example Valerianella;
• - Chenopodiaceae, for example Beta;
• - Cucurbitaceae, for example Cucumis;
• - Fabaceae, for example Glycine. Lens, Pisum, Trifolium, Vicia, Lupinus;
• - Lamiaceae, for example Ajuga, Lavendula;
• - Ranunculaceae, for example Delphinium;
• - Rosaceae, for example Fragaria, Filipendula;
• - Solanaceae, for example Lycopersicum, Solanum.

Very particularly preferred is a method according to the invention, where the plant seeds are seeds of the plants listed in Tables 1 and 2.

The different components of the test composition have different meanings in the method according to the invention. According to the invention, the solvent must comprise water, since the presence of water is essential for the later desired functionality. At least in principle it is possible for other solvents to be present in addition to water, so that the test composition as a whole comprises an aqueous solvent. In practice, however, the inventors consider it to be preferable, among other things with a view to environmental compatibility, the seed-preserving nature of the process and the overall costs, if the solvent used in the test composition is 95% or more, preferably 98% or more, particularly preferably 99%. or more, very particularly preferably essentially completely, consists of water, based on the mass of the solvent, which can advantageously also prevent any further solvent components from negatively influencing the activity of the fermenting microorganism and/or the turnover behavior of the redox indicator. Accordingly, a method according to the invention is preferred, where the water is distilled or demineralized water.

In the prior art, test compositions are sometimes referred to as test solutions. In accordance with the expert understanding, the test composition will not in all cases be a solution in the narrower sense, i.e. H. be an essentially homogeneous solution. The person skilled in the art understands that the term test composition is more suitable because, in addition to test solutions, it also includes those liquid systems in which undissolved particles may be present, for example as a result of an incompletely dissolved fermented microorganism, so that these systems are more likely to be referred to as test suspensions.

The test composition to be used includes a two-stage redox indicator, which can indicate a change in the redox potential of the test composition. Redox indicators themselves and their functionality are known to those skilled in the art from the prior art, as are two-stage redox indicators. These two-stage redox indicators have two transitions in which the absorption properties, and thus in most cases also the visually perceptible color, change as a result of a reduction or oxidation reaction. Two-stage redox indicators therefore have three different redox states between which the two redox transitions lie.

According to the inventors' assessment, the concept underlying the method according to the invention can in principle be implemented with any two-stage redox indicator. However, because of the application already known from the prior art, the advantageous risk profile and the wavelength ranges of absorption of the various redox states that are favorable for the instrumental evaluation, resazurin in particular is predestined for use in the method according to the invention and for all embodiments as a two-stage redox indicator preferred. Resazurin is well known to those skilled in the art from the prior art and, like other redox indicators, is commercially available from various manufacturers. A method according to the invention is preferred, wherein the two-stage redox indicator is selected from the group consisting of dyes with a phenoxazin-3-one skeleton, for example resazurin, where the two-stage redox indicator is preferably resazurin.

The test composition includes a fermenting microorganism as a further component. The term “fermenting microorganism” is clear to the person skilled in the art and refers to sol che microorganisms, such as bacteria and fungi, which are able to convert organic substances, in particular glucose, microbially or enzymatically as part of the so-called fermentation. According to the inventors' assessment, single-celled fungi, which are known to the person skilled in the art, for example in the form of yeast, such as baker's yeast, are particularly suitable as fermenting microorganisms. Accordingly, preference is given to a method according to the invention, wherein the fermenting microorganism is selected from the group consisting of unicellular fungi, preferably is selected from the group consisting of yeasts, particularly preferably is selected from the group consisting of Saccharomyces cerevisiae and Saccharomyces bayanus. With regard to the production of the solution, preference is given to a method according to the invention, the test composition being prepared in process step b), the fermenting microorganism being used as a cold-treated microorganism, preferably as a freeze-dried microorganism.

In the method according to the invention, the test composition, at least in the broadest defined form, has the same task that is known from the prior art, whereas the preferred test compositions according to the invention disclosed below also advantageously fulfill additional functions. Without wishing to be bound to this theory, the inventors assume that the processes taking place in the method according to the invention can be described as follows. When dry, selectively permeable membranes in plant seeds are not functional. After contact with water, the plant seeds begin to absorb water. Depending on the physiological constitution, the selectively permeable membrane is either completely or partially reconstituted, so that the leaching of storage substances such as carbohydrates, proteins or fats varies. If the semen is of good constitution, it controls leaching so that there is only a low concentration of these organic substances in the test composition. In contrast, the reconstitution of the selectively permeable membrane hardly works at all in dead plant seeds. This results in a strong leaching of storage substances, which are present in increased concentrations in the test composition. Accordingly, the condition of the plant seeds correlates with how much organic substances are available to the fermenting microorganism in the test composition during incubation. This means that depending on the vitality of the plant seeds, there is a different activity of the microorganisms in the test composition. The redox indicator now serves to make the different activities of the microfermenting microorganisms detectable or even visible. This can be illustrated using the example of resazurin. Resazurin is a two-stage redox indicator that is colored dark blue-violet when fully oxidized. As a result of a first reduction, the color changes towards pink (irreversible), whereas as a result of a second reduction a colorless form is obtained (reversible). In other words, it is a method according to the invention, in which the fermenting microorganism can change the redox potential of the test composition in the course of the fermentation of fermentable compounds, and/or in which the test composition is designed to ensure that the fermentation of fermentable compounds by the fermenting Microorganism in the test composition first triggers a first color change and then a second color change of the two-stage redox indicator, or a first reduction and then a second reduction.

The inventors have recognized that in order to achieve particularly advantageous estimation results, it is expedient to adapt the composition of the test composition to the plant seeds to be examined in the method according to the invention, in particular with regard to the concentration of the redox indicator and the fermenting microorganism. In this respect, the inventors suggest that in a kit according to the invention, as disclosed below, in particular plant seed-specific production instructions for producing a corresponding test composition from a prepared solid, for example a powder, by mixing with water or an aqueous solvent can be provided in order to produce a specific To allow mixing of the components. In this respect, the inventors have succeeded in identifying fundamentally preferred areas with which, in the inventors' opinion, particularly advantageous test compositions can be obtained which can be used for a wide range of plant seeds even without plant seed-specific production. Namely, preference is given to a method according to the invention, wherein the mass fraction of water in the test composition is 70% or more, preferably 80% or more, particularly preferably 90% or more, very particularly preferably 95% or more, particularly preferably 99% or more, based on the mass of the test composition. Additionally or alternatively, a method according to the invention is preferred, wherein the mass fraction of the two-stage redox indicator in the test composition is in the range from 0.00001 to 5%, preferably in the range from 0.001 to 0.5%, particularly preferably in the range from 0.01 to 0.05%, based on the mass of the test composition. Additionally or alternatively, a method according to the invention is preferred, wherein the mass fraction of the fermenting microorganism in the Test composition is in the range from 0.01 to 10%, preferably in the range from 0.05 to 5%, particularly preferably in the range from 0.1 to 0.5%, based on the mass of the test composition.

In process step c), the plant seed portions are contacted with a test volume of test composition. In accordance with professional understanding, this means that each plant seed is contacted with a separate portion of the test composition. The contacting can expediently take place in a suitable vessel, and in view of the usually large number of plant seeds that are usually examined at the same time in the method according to the invention, the use of so-called multiwell plates is particularly expedient. In many cases, a method according to the invention is expedient, wherein the plant seed portions are contacted with a test volume of test composition in each case in a test recess from a plurality of test recesses of a first test plate, so that the separate test systems are obtained in the different test recesses of the first test plate.

As explained above, in order to avoid signs of aging in the test composition, which could adversely affect the result of the method according to the invention, it is advisable to prepare the test composition as soon as possible before contacting the plant seed portions. In order to achieve a particularly good result in this respect, the inventors recommend that the fermenting microorganism should be used as a cold-treated microorganism before mixing with the solvent, as is also disclosed above. In order not to increase the activity of the fermenting microorganism too early, the inventors recommend using relatively cold water when preparing the test composition. In an advantageous further development, this can be carried out to such an extent that the temperature of the test composition is kept as low as possible until the moment the plant seeds come into contact in order to prevent a premature, undesirable increase in the activity of the fermenting microorganisms. In comparison to a method that prepares the solution at room temperature, this can advantageously prevent any redox processes in the solution produced from causing initial changes in the redox indicator, which can falsify the results of the subsequent optical measurements. Accordingly, preference is given to a method according to the invention, wherein the test composition is produced in method step b), the water during production having a temperature of 8 ° C or less, preferably 7 ° C or less, preferably 6 ° C or less, particularly preferably 5 °C or less. Particularly preferred in this respect is a method according to the invention, wherein the test composition has a temperature of 8 ° C or less, preferably 7 ° C or less, preferably 6 ° C or less, particularly preferably 5 ° C or less, when contacting the plant seed portions.

By contacting the different plant seed portions with a respective part of the test composition, a large number of subunits are obtained, which are referred to as test systems in the context of the present invention for the purpose of clear identification. These test systems therefore include test composition and a plant seed portion. The person skilled in the art understands that the volume of the test composition added in each case will expediently depend on the size of the plant seeds and/or the number of plant seeds in the plant seed portion. For individual, small plant seeds, the person skilled in the art expediently chooses a lower volume than for larger plant seed portions of relatively large seeds. The person skilled in the art also understands that the efficiency of the transfer of organic substances from a plant seed into the test composition, which are identified via the combination of the fermenting microorganism and the two-stage redox indicator, is usually largely determined by the contact surface between the plant seed and the test composition will depend. Even if it is in principle possible to use small test volumes of test composition that only contact parts of the plant seeds, the inventors believe that this is not the preferred procedure. At the same time, large volumes of test composition also mean that a large amount of redox indicator is present and large parts of the redox indicator may not be able to turn over, even in dead seeds. According to the inventors, it is particularly preferred if the test volume of test solution in the test systems is in the range from 1*V _s to 500*V _s , preferably in the range from 2*V _s to 250*V _s , particularly preferably in the range from 3*V _s to 125 *V _s , where V _s is the combined volume of plant seeds in the plant seed portion. Additionally or alternatively, a method according to the invention is preferred, wherein the plant seed portions are each contacted with a test volume of test solution in the range from 0.1 to 500,000 µL, preferably in the range from 1 to 50,000 µL, particularly preferably in the range from 10 to 5000 µL .

In order to increase the efficiency of the method according to the invention, the inventors of the present invention suggest that measures be taken to improve the wetting of the plant seeds should, as this increases the interface area and enables a more efficient transfer of organic substances from the plant seeds into the test composition. In particular, the time required to carry out the method according to the invention can be significantly reduced by appropriate measures.

As a first, less preferred option for setting advantageous wetting, the inventors suggest that the test systems can be centrifuged in order to achieve advantageous wetting. This embodiment is therefore a method according to the invention, wherein the contacting of the plant seed portions with a test volume of test composition before incubation is supported by a mechanical treatment step, preferably by submerging the plant seeds or by centrifuging the test systems, particularly preferably by centrifuging the test systems .

However, the inventors consider the use of a specific surfactant, a so-called detergent, to be particularly advantageous and preferred for essentially all embodiments of the method according to the invention, with which, in contrast to the method known from the prior art, an advantageous wetting of the plant seed portions can be achieved. This achieves a significant increase in the efficiency of the process, especially with regard to the time required, without the need for centrifugation, for example. In order to implement this preferred embodiment, the inventors consider it essential that not just any surfactant should be used, but rather that surfactants should be used which do not or at least only slightly negatively influence the bioactivity of the fermenting microorganism. Particularly preferred is therefore a method according to the invention, wherein the test composition additionally comprises one or more surfactant compounds that are biocompatible with the fermenting microorganism, preferably in a combined mass fraction in the range from 0.001 to 25%, preferably in the range from 0.01 to 5%, especially preferably in the range from 0.1 to 0.5%, based on the mass of the test solution.

The term biocompatible is clear to the person skilled in the art and expresses that the activity of a microorganism in the presence of the surfactant compounds is less than 5%, preferably less than 1%, particularly preferably less than 0.1%, particularly preferably essentially not at all , is reduced. Many suppliers of commercially available detergents point out in the documentation to what extent the surfactant compounds offered are compatible with microorganisms, i.e. H. biocompatible, are or can provide relevant information upon request. Additionally or alternatively, the person skilled in the art can also use comparatively simple tests with the fermenting microorganism he is using to determine whether a surfactant compound in his possession is sufficiently biocompatible. According to the inventors' assessment, non-ionic surfactants in particular are particularly suitable for use in the process according to the invention, with polysiloxane-based polymers in particular having led to excellent results in the inventors' own experiments. Particularly advantageous results were achieved with trisiloxane-based non-ionic surfactants. Corresponding non-ionic surfactants are commercially available from various manufacturers, for example under the trade name Break-Thru, for example as Break-Thru SD260, from Evonic Operations GmbH. A process according to the invention is particularly preferred, wherein the surfactant compound is selected from the group consisting of non-ionic surfactants, preferably is selected from the group consisting of polysiloxane copolymers, particularly preferably is selected from the group consisting of polyether Polyalkylsiloxane copolymers.

In the method according to the invention, the test systems obtained are then incubated. The “incubation” step is clear to those skilled in the art and refers to the development of the test systems for a specified period of time, usually at an elevated temperature. In the method according to the invention, the time of incubation serves in particular to enable the organic substances to escape from the plant seeds and the conversion of these substances by the fermenting microorganism. The inventors have succeeded in identifying particularly advantageous conditions for incubation, with which good results can be obtained for most typical plant seeds of industrially relevant crops, with the trade-off between time and energy efficiency on the one hand and the most reliable estimate possible the germination properties on the other hand are solved particularly advantageously in these configurations. A method according to the invention is therefore preferred, with the incubation taking place for a time in the range from 0.5 to 24 hours, preferably in the range from 1 to 12 hours, particularly preferably in the range from 1.5 to 8 hours. Additionally or alternatively, a method according to the invention is also preferred, with the incubation taking place in the absence of light. Additionally or alternatively, a method according to the invention is also preferred, wherein incubation at a temperature in Range from 6 to 40 ° C, preferably in the range from 8 to 37 ° C, particularly preferably in the range from 10 to 35 ° C. In principle, preference is given to a method according to the invention, in which the incubation takes place for a predetermined time depending on the type of plant seeds, the predetermined time preferably being specified via a plant seed-specific instruction of a kit according to the invention.

The person skilled in the art understands that at the end of the incubation a test composition is obtained in the incubated test systems in which part of the two-stage redox indicator has been converted once or twice and that these test compositions of the test systems are measured in the further course of the method according to the invention.

This measurement of the test compositions of the incubated test systems is carried out in process step d), using an optical measurement method.

Suitable optical measurement methods are known to those skilled in the art based on their specialist knowledge, with suitable optical measurement devices, in particular powerful digital designs, being commercially available from various providers. In view of the measurement data to be recorded, a method according to the invention is relevant for essentially all embodiments, wherein the optical measurement method is an absorption measurement method, preferably a transmission measurement method. Due to the possibility of carrying out data processing steps of the method according to the invention on the optical measuring device, a method according to the invention is preferred, wherein the optical measuring device comprises the electronic data processing device.

The person skilled in the art understands that as a result of the change or changes of the two-stage redox indicator, the wavelength of the absorption maximum of the test composition shifts, which can be perceived by the person skilled in the art as a color change, at least in the case of changes in which the changes are in the visible light range. The person skilled in the art therefore understands that in the method according to the invention reference is made to a quantity which correlates with the absorption properties of the respective test composition from the incubated test systems.

It is clear to the person skilled in the art that the simplest and most direct absorption properties can consist of the directly measured absorption values. At the same time, however, it may be expedient to determine parameters and/or values derived therefrom instead of the directly determined absorption properties and/or to evaluate them as part of the subsequent evaluation. In particular, the later evaluation can be carried out using normalized or normalized absorption values, which are corrected, for example, by calculation operations in the light of absorption properties determined on blind systems, reference systems and/or control systems, as is also described below. In accordance with the expert understanding, an absorption data set is obtained for each test system, which includes information about the absorption properties of the respective test composition, whereby these absorption properties can also include values derived therefrom in addition to the directly determined absorption values, as described above.

Unlike the method known from the prior art, the absorption properties of the respective incubated test compositions are not only measured at 570 nm. Rather, the optical absorption properties of the respective test composition are determined at a first wavelength λ ₁ and a second wavelength λ ₂ , whereby, according to the invention, these wavelengths must differ by at least 10 nm. In other words, it is a method according to the invention, wherein the measurement is carried out with an optical measuring device, preferably an optical photometer, the optical measuring device being set up to measure the optical absorption properties of the test composition for electromagnetic radiation of at least a first wavelength λ ₁ and a second wavelength λ ₂ , where λ ₁ and λ ₂ differ by 10 nm or more.

• i) die Absorptionswerte der jeweiligen Testzusammensetzungen bei der ersten Wellenlänge λ₁ und der zweiten Wellenlänge λ₂, oder
• ii) aus diesen Absorptionswerten abgeleitete Werte,

umfassen, wobei die aus den Absorptionswerten abgeleiteten Werte bevorzugt durch eine oder mehrere Rechenoperationen erhalten werden, insbesondere durch Wertnormierung oder Wertkorrektur, beispielsweise in Abhängigkeit von Absorptionswerten die an Kontrollsystemen, Referenzsystemen oder Blindsystemen bestimmt wurden.In light of the above, an example is a method according to the invention, wherein the absorption properties are at least

• i) the absorption values of the respective test compositions at the first wavelength λ ₁ and the second wavelength λ ₂ , or
• ii) values derived from these absorption values,

include, wherein the values derived from the absorption values are preferably obtained by one or more calculation operations, in particular by value normalization or value correction, for example depending on absorption values that were determined on control systems, reference systems or blind systems.

The absorption properties of the respective test compositions for electromagnetic radiation of at least a first wavelength λ ₁ and a second wavelength λ ₂ can, for example, be determined selectively, only at the corresponding wavelengths, for example when using essentially monochromatic radiation sources or by using suitable filters. Alternatively, spectra can also be recorded in a broader wavelength range and the first and second wavelengths can be read out at the specific wavelengths.

In addition to the specific form of evaluation of the data sets, the advantages of the method according to the invention result from the fact that the absorption data sets intended for the various test systems include information about the absorption behavior at two different wavelengths that have a certain minimum distance from one another. The total absorption spectra of the incubated test compositions can be viewed in simplified terms as a superposition, ie as a superposition, of the absorption spectra weighted with the mass fractions of the components contained therein, in particular of the different states of the redox indicator. The minimum distance between the first and second wavelengths thus determines the absorption properties of the solution at two wavelengths spaced apart from one another, at which the different, differently oxidized states of the two-stage redox indicator contribute to the combined absorption spectrum to a different extent and a different ratio. The inventors view this information content of the absorption data sets obtained as central to the subsequent evaluation using machine learning. In their own experiments, the inventors have identified a distance of 10 nm as a sensible lower limit with which meaningful results can still be obtained with a reasonable resource requirement before the changing contributions of the redox states no longer differ sufficiently. However, the inventors suggest that in most cases better estimation results can be achieved with larger wavelength differences, since the relative contribution of the redox states of the redox indicator in these cases regularly deviates more strongly in view of typical absorption spectra. A method according to the invention is therefore preferred, wherein the first wavelength λ ₁ and the second wavelength λ ₂ differ by 15 nm or more, preferably by 20 nm or more, particularly preferably by 25 nm or more.

Even if it were fundamentally possible to measure the absorption behavior at three or more different wavelengths, and additionally, for example, to determine the absorption properties at a third wavelength λ ₃ and a fourth wavelength λ ₄ , to include the corresponding information in the absorption data sets and to use this in the In the opinion of the inventors, this is not preferred in the later computer-aided estimation. The person skilled in the art understands that each additionally measured wavelength can potentially increase the estimation accuracy, but that this would involve an increase in the amount of equipment required for the measurement and an increase in the required storage and computing capacity. In the inventors' eyes, the excellent estimation quality that can already be achieved with two wavelengths makes it unnecessary to measure further wavelengths, so that the additional effort is not considered justified, especially if the absorption properties are determined at wavelengths that are close to the respective absorption maximum of the relevant redox -States of the redox indicator are, for example, at 570 nm and 600 nm for resazurin.

In practice, the efficient choice of the first and second wavelength will essentially be determined by the choice of the two-stage redox indicator. In this respect, the inventors suggest that, provided the minimum distance described above is maintained, the first wavelength is in the range from (m ₁ -20) nm to (m ₁ +20) nm, preferably in the range from (m ₁ -10) nm to (m ₁ +10) nm, where m ₁ is the wavelength of the absorption maximum of a redox state of the two-stage redox indicator, and / or that the second wavelength is in the range from (m ₂ -20) nm to (m ₂ +20) nm, preferably in the range from (m ₂ -10) nm to (m ₂ +10) nm, where m ₂ is the wavelength of the absorption maximum of a redox state of the two-stage redox indicator.

With regard to the particularly preferred two-stage redox indicator resazurin, the inventors suggest wavelength ranges with which excellent results could be obtained in their own experiments and which result in absorption data sets that are used in the following evaluation can be evaluated particularly well. Namely, a method according to the invention is preferred, wherein the first wavelength λ ₁ is in the range from 585 to 630 nm, preferably in the range from 590 to 620 nm, particularly preferably in the range from 595 to 610 nm. Additionally or alternatively, preferably additionally, a method according to the invention is preferred, wherein the second wavelength λ ₂ is in the range from 540 to 585 nm, preferably in the range from 550 to 580 nm, particularly preferably in the range from 560 to 575 nm.

According to the inventors' assessment, it is at least theoretically possible to subject the test systems directly to a transmission measurement, i.e. H. without first removing the plant seed portion. In practice, however, this can result in an avoidable deterioration of the absorption measurement data obtained, so that the inventors rather suggest that subvolumes of test composition should be removed from the incubated test systems and fed to the optical measurement method in order to enable the process to be carried out as efficiently as possible. In this respect, preference is given to a method according to the invention, wherein the measured test composition of the incubated test systems is separated from the plant seed portions before measurement, with a subvolume of test composition, particularly preferably a subvolume in the range of 50 to 150 µL, being removed from the test systems in each case to measure this, with the various subvolumes preferably being transferred into the test recesses of a second transparent test plate.

Before the subsequent evaluation of the absorption data sets obtained to estimate the germination properties is explained in more detail below, it is expedient to address two aspects that relate in particular to process steps a) to d). The person skilled in the art readily understands that a subsequent correlation of the determined absorption properties of the test systems with the germination properties of plant seeds for the purpose of estimating the germination properties is easier and requires less computing capacity, the lower the number of other disturbing factors and deviations.

For the person skilled in the art, this means that the absorption data sets of the test systems should naturally be generated under the same conditions as possible. Such an approach corresponds to the natural approach of the expert when designing a series of measurements. This means, for example, that plant seeds of the same type are preferably used, plant seed portions of the same size are formed, essentially the same volumes of test composition are used, the test composition is composed as uniformly as possible within the method, the method of contacting the plant seed portion with the test volume is as similar as possible and the Absorption properties can be determined, for example, under the same conditions as possible, in particular with the same optical measuring devices and measuring methods.

Theoretically, a large number of deviations, especially if they are comparatively small, can be compensated relatively well in the subsequent evaluation by resorting to machine learning, at least if in return the available computing capacity is increased and a correspondingly powerful estimation module is used. However, in accordance with the expert understanding, a method according to the invention is expedient for all embodiments, in which a large number of plant seed portions of similar size of similar plant seeds are contacted with essentially equal volumes of a substantially identical test composition and incubated under essentially the same conditions, the test compositions being the same Test systems incubated in this way are measured using the same optical measuring method under the same measuring conditions to determine the optical absorption properties of the respective test compositions. In this respect, a method according to the invention is particularly preferred, with each test system containing the same number of plant seeds and essentially the same amount of test composition. A method according to the invention is particularly preferred, wherein the test compositions of the incubated test systems are measured under identical conditions.

Another factor that the inventors believe is particularly advantageous in terms of efficient process management is the use of suitable comparison systems that enable efficient evaluation of the plant seed portions measured together.

According to the inventors' assessment, it is initially expedient to provide so-called blind systems in particular, which do not include plant seed portions or test compositions, but only include the solvent used in the test composition. These blind systems can be used in particular to correct the optical absorption properties determined in process step d) in order to correct the influence of the solvent and, if necessary, the sample carrier. A method according to the invention is therefore preferred, wherein in method step c), in addition to the large number of test systems, one or more blind systems are generated, incubated with the test system and measured in method step d), the blind system comprising exclusively the solvent of the test composition, in particular water. Particular preference is given to a method according to the invention, wherein the optical absorption properties of the respective test compositions determined in method step d) are corrected as a function of the optical absorption properties of the one or more blind systems, preferably by subtracting the blank values.

In addition, the inventors suggest that control systems can also be processed in which, instead of a plant seed portion, a known amount of a fermentable compound is presented in a controlled manner in order to check that the test compositions are sufficiently functional, i.e. H. to check that the fermenting microorganism in particular is active and can cause a change in a redox indicator. Accordingly, preference is given to a method according to the invention, wherein in method step c), in addition to the large number of test systems, one or more control systems are generated, incubated with the test system and measured in method step d), the control systems containing a fermentable compound, preferably a carbohydrate compound Test composition include.

The inventors consider the use of these control systems to be particularly preferred because the absorption properties determined on the respective test compositions of the test systems can be standardized using the absorption properties determined on the control systems. In practice, the observed biological processes are usually limited in their reproducibility, and various environmental factors can affect the activity of the microorganisms and the intensity of leaching. In practice, through the use of control systems, both biotic fluctuations, such as in the actual concentration of the microorganisms used or their motility, i.e. fitness, as well as abiotic fluctuations, such as temperature fluctuations or different water qualities between measurements on different days, and / or in different locations, and/or in different weather conditions, so that comparable absorption information can be obtained under changing conditions. The normalization preferred for essentially all embodiments allows these factors to be at least partially and in many cases even almost largely ignored, which advantageously makes it easier to carry out the method according to the invention outside of highly controlled laboratory conditions, so that the implementation of a decentralized Procedure, for example with an absorption measurement on the farmer, is much easier. The normalization can be carried out, for example, based on the measured values at the wavelength that corresponds to the fully oxidized form of the two-stage redox indicators. Particularly preferred in any case is a method according to the invention, wherein the optical absorption properties of the respective test compositions determined in method step d) are normalized as a function of the optical absorption properties of the one or more control systems.

In addition to blind systems and control systems, the inventors suggest that reference systems that exclusively include the test composition can also or alternatively be used. In terms of their color and absorption behavior, these reference systems correspond to the initial state in which the fermentation has not yet caused a change in the redox indicators. In this way, the reference systems are particularly suitable for determining suitable incubation conditions. If there is sufficient color contrast between the control system in which fermentation takes place and the reference system, it can often be seen with the naked eye that the incubation has been carried out for a sufficiently long time. A method according to the invention is therefore preferred, wherein in method step c), in addition to the large number of test systems, one or more reference systems are generated, incubated with the test system and measured in method step d), the reference systems exclusively comprising test composition. A method according to the invention is also preferred, in which the test systems are incubated until a predetermined color contrast results between the control system and the reference system.

In process step e), the germination properties of the plant seeds are estimated, which is disclosed in detail below.

• f) Berechnen einer mittleren Keimungseigenschaftsprognose durch Mittelwertbildung aus den für die Pflanzensamenportionen abgeschätzten Keimungseigenschaften.

The isolated information about the estimated germination properties of a plant seed or a plant seed portion is usually of little relevance in practice. The added value of the method according to the invention lies in particular in the fact that a larger number of plant seeds, which are suitable for a larger ßere seed batch are representative, can each be estimated with regard to their germination properties in order to derive an average germination properties forecast for the seed batch, which can be used as a characteristic value for the seed batch. A method according to the invention is therefore initially preferred, the number of plant seed portions and the number of test systems being 20 or more, preferably 40 or more, particularly preferably 60 or more, very particularly preferably 80 or more. Accordingly, a method according to the invention is also preferred, additionally comprising the method step:

• f) Calculating an average germination property prediction by averaging the germination properties estimated for the plant seed portions.

• g) Ausgeben der abgeschätzten Keimungseigenschaften oder der berechneten mittleren Keimungseigenschaftsprognose, wobei das Ausgeben bevorzugt über eine Datenschnittstelle oder elektronische Anzeigevorrichtung der elektronischen Datenverarbeitungsvorrichtung erfolgt. Mit Blick auf eine einfache Handhabbarkeit der ausgegebenen Daten ist dabei die Ausgabe in einer leicht verständlichen graphischen Darstellung bevorzugt, beispielsweise in einem sogenannten Box-Plot, sodass ein direkter und intuitiver Abgleich der zur Verfügung stehenden Daten besonders einfach ermöglicht wird.

In most cases it will be expedient to output the information obtained in method step e) in a suitable manner, for example via a display, although output via an electronic interface for the purpose of further data processing is also conceivable. A method according to the invention is therefore preferred, additionally comprising the method step:

• g) outputting the estimated germination properties or the calculated average germination property prediction, the output preferably taking place via a data interface or electronic display device of the electronic data processing device. With a view to making the output data easy to handle, output in an easily understandable graphical representation is preferred, for example in a so-called box plot, so that a direct and intuitive comparison of the available data is particularly easy.

In the method according to the invention, the absorption data sets assigned to the test systems are evaluated in a computer-assisted manner using the concept of machine learning (sometimes also referred to as “artificial intelligence” or “AI-based”) to estimate the germination properties. Here, the absorption data sets determined on the various test systems as described above, which include information about the absorption properties of the respective test compositions at the first wavelength λ ₁ and the second wavelength λ ₂ , are given as input from the electronic data processing device into an estimation module based on machine learning, which makes the actual estimate and outputs corresponding estimated information about the germination properties.

The electronic data processing device can, for example, be a separate computer at the end user, which is connected, for example, to the optical measuring device. However, as part of an integrated solution, it can also be an electronic data processing device that is part of the optical measuring device. In particularly preferred embodiments, however, the electronic data processing device is a central electronic data processing device, for example a server as part of a cloud-based evaluation, which can be connected in a network, for example, to a large number of optical measuring devices.

In the context of the present invention, the designation of the module based on machine learning as an estimation module serves to conveniently name and identify the module via its function, with its suitability for estimating the germination properties from the absorption data sets assigned to the test systems resulting from the training of the estimation module. The estimation module can, for example, be part of a more comprehensive software and is present on the storage unit of the electronic data processing device, the term storage unit denoting a memory that can be accessed by the electronic data processing device and which does not necessarily have to be physically connected to the electronic data processing device, but also via a wireless one, for example Communication network can be accessed.

The concept of machine learning itself as well as suitable algorithms for machine learning are fundamentally familiar to the person skilled in the art based on their specialist knowledge. Modules or computer program products based on machine learning, which can be adapted to the requirements of the present invention using the training data sets specified here, are commercially available from numerous manufacturers or developers. An example is a method according to the invention, wherein the estimation module is based on a machine learning algorithm, which is selected from the group consisting of supervised learning algorithms, preferably selected from the group consisting of supervised learning algorithms for solving regression problems, particularly preferably selected is from the group consisting of artificial neural networks, and/or wherein the estimation module is obtained by applying a machine learning algorithm to the set of training data, the algorithm being selected from the group consisting of supervised learning algorithms, preferably selected from the group consisting of supervised learning algorithms Learning to solve regression problems is particularly preferably selected from the group consisting of artificial neural networks.

• - R Core Team (2021). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL: https://www.R-project.org/.
• - Mlr3: Lang M, Binder M, Richter J, Schratz P, Pfisterer F, Coors S, Au Q, Casalicchio G, Kotthoff L, Bischl B (2019). „mlr3: A modern object-oriented machine learning framework in R.“ Journal of Open Source Software, doi: 10.21105/joss.01903 (URL: https://doi.org/10.21105/joss.01903), URL: https://joss.theoj.org/papers/1 0.211 05/joss.01903.
• - Mlr3viz: Michel Lang, Patrick Schratz, Raphael Sonabend, Marc Becker and Jakob Richter (2021). mlr3viz: Visualizations for ‚mlr3‘. R package version 0.5.7. https://CRAN. R-project.org/package=mlr3viz.
• - Tidyverse: Wickham et al., (2019). Welcome to the tidyverse. Journal of Open Source Software, 4(43) , 1686, https://doi.org/10.21105/joss.01686.

The inventors refer in particular to the following publications, which are considered particularly helpful:

• - R Core Team (2021). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL: https://www.R-project.org/.
• - Mlr3: Lang M, Binder M, Richter J, Schratz P, Pfisterer F, Coors S, Au Q, Casalicchio G, Kotthoff L, Bischl B (2019). “mlr3: A modern object-oriented machine learning framework in R.” Journal of Open Source Software, doi: 10.21105/joss.01903 (URL: https://doi.org/10.21105/joss.01903), URL: https: //joss.theoj.org/papers/1 0.211 05/joss.01903.
• - Mlr3viz: Michel Lang, Patrick Schratz, Raphael Sonabend, Marc Becker and Jakob Richter (2021). mlr3viz: Visualizations for 'mlr3'. R package version 0.5.7. https://CRAN. R-project.org/package=mlr3viz.
• - Tidyverse: Wickham et al., (2019). Welcome to the tidyverse. Journal of Open Source Software, 4(43) , 1686, https://doi.org/10.21105/joss.01686.

To obtain the estimation module (i.e. the “training”), the person skilled in the art uses a training set of training data, which are corresponding absorption data sets of plant seed portions of plant seeds with known germination properties, which are referred to in the context of the invention as training absorption data sets of training plant seed portions, so that the identification module can be trained using supervised learning to enable the desired functionality.

Fortunately, obtaining a suitable training set does not pose a problem for the expert in practice, since carrying out germination tests, which are usually based on the ISTA specifications, is day-to-day business for the expert in practice and the information required for “supervised learning” is also available in many cases Cases are binary (i.e. germinates/does not germinate). Regardless of the fact that the forecast quality can be increased if necessary through suitable measures, as will be described below using the practical example, the person skilled in the art only needs to generate corresponding absorption data sets in some of the germination tests that they already carry out, which in turn is done with process steps a) to d). can.

The following example uses an example to illustrate how an exemplary set of training data can be generated. In addition, an exemplary set of actually measured training data is disclosed below in Table 3, which gives the person skilled in the art an impression of a suitable format of the training data using the example of rapeseed and can also act as an initial basis for training an exemplary estimation module for testing purposes.

1. A. Zunächst wird das erfindungsgemäße Verfahren im Rahmen eines eher groben „Screenings“ durchgeführt, um zu bewerten, unter welchen Bedingungen eine zielführend Differenzierung der Absorptionseigenschaften der Testsysteme zu finden ist (Variation Volumen, Konzentration RedOx-Indikator, Inkubationsdauer, Inkubationstemperatur, etc.).
2. B. Wenn eine hinreichende Differenzierung erreicht wurde, werden größere Mengen von Pflanzensamen den Verfahrensschritten a) bis d) unterzogen, wobei die Inkubation unter den gemäß Punkt A ermittelten Bedingungen erfolgt. Hierbei werden, wie vorstehend beschrieben, Blindsysteme, Kontrollsysteme und Referenzsysteme, eingesetzt, um hintergrundbereinigte und normierte Informationen über die Absorptionseigenschaften der jeweiligen Testzusammensetzungen bei der ersten Wellenlänge λ₁ und der zweiten Wellenlänge λ₂ zu erhalten.
3. C. Anschließend werden die zuvor untersuchten Pflanzensamen so nach ISTA-Kriterien ausgesät, dass eine Zuordnung der aufgenommenen Absorptionsdatensätze zu den ausgesäten Pflanzensamen möglich bleibt, wobei (je nach dem Ziel des späteren Trainings) beispielsweise erfasst werden kann, welche Pflanzensamen physiologisch keimen, wie lange es zur physiologischen Keimung dauert, und/oder welche Pflanzensamen einen „normalen Keimling“ ausbilden.
4. D. Die Inkubation in der Testzusammensetzung stellt für die Pflanzensamen trotz des zerstörungsfreien Charakters des Verfahrens Stress dar. Insbesondere ein Sauerstoffmangel bei vollständiger Bedeckung mit Testzusammensetzung kann die Vitalität der getesteten Pflanzensamen beeinflussen. Dieser Einfluss der Inkubation auf die Keimungseigenschaften ist bei der Zusammenstellung der Trainingsdaten nicht zu vermeiden. Die Erfinder haben jedoch erkannt, dass es zur Verbesserung der Abschätzungsqualität möglich ist, diesen Effekt auszugleichen, indem die in Punkt C erhaltenen Informationen abschließend auf eine nicht-inkubierte, d.h. „normale“, Aussaat nach ISTA kalibriert werden. Dazu wird dieselbe Anzahl Pflanzensamen ohne vorherige Inkubation nach den gleichen ISTA-Kriterien ausgesät, dieselben Keimungseigenschaften erfasst und anschließend eine Kalibrierfunktion ermittelt.

The inventors suggest that a suitable set of training data for a type of plant seed can be generated, for example, as follows:

1. A. First, the method according to the invention is carried out as part of a rather rough “screening” in order to evaluate the conditions under which a targeted differentiation of the absorption properties of the test systems can be found (variation volume, concentration of RedOx indicator, incubation duration, incubation temperature, etc.) .
2. B. If sufficient differentiation has been achieved, larger quantities of plant seeds are subjected to process steps a) to d), with incubation taking place under the conditions determined in accordance with point A. Here, as described above, blind systems, control systems and reference systems are used to obtain background-corrected and standardized information about the absorption properties of the respective test compositions at the first wavelength λ ₁ and the second wavelength λ ₂ .
3. C. The previously examined plant seeds are then sown according to ISTA criteria in such a way that it remains possible to assign the recorded absorption data sets to the sown plant seeds, whereby (depending on the goal of the later training) it can be recorded, for example, which ones Plant seeds germinate physiologically, how long it takes for physiological germination, and/or which plant seeds form a “normal seedling”.
4. D. Incubation in the test composition represents stress for the plant seeds despite the non-destructive nature of the process. In particular, a lack of oxygen when completely covered with the test composition can influence the vitality of the tested plant seeds. This influence of incubation on germination properties cannot be avoided when compiling the training data. However, the inventors have recognized that, in order to improve the quality of the estimation, it is possible to compensate for this effect by finally calibrating the information obtained in point C to a non-incubated, ie "normal", sowing according to ISTA. To do this, the same number of plant seeds are sown without prior incubation according to the same ISTA criteria, the same germination properties are recorded and a calibration function is then determined.

The inventors usually recommend a size of at least 10 “batches” of different batches and qualities for the training data sets, each with at least 400 seeds, although in many cases at least simpler estimation modules can also be obtained with significantly smaller training data sets.

With the training data sets obtained, regression models can be trained in the usual way using machine learning to obtain the estimation module, which are trained using the normalized absorption data sets of at least two wavelengths, the corresponding normalization factor and the recorded germination properties (e.g. physiologically germinated yes/no). The quality of the models can, for example, be checked directly with a previously separated portion of the training data (e.g. approx. 20%), for example in order to determine the mean square error and/or the maximum error .: “maximum error”) of the obtained estimation module and decide whether further training is necessary.

The above explanations result in particularly preferred embodiments of the method according to the invention, which can be implemented individually or in combination of two or more features.

A method according to the invention is preferred, wherein the set of training data comprises 3000 or more, preferably 4000 or more, particularly preferably 5000 or more, training absorption data sets of training plant seed portions of plant seeds with known germination properties.

In principle, preference is given to a method according to the invention, wherein the set of training data is obtained by carrying out method steps a) to d) of a method according to the invention, preferably a substantially identically executed method according to the invention, for a plurality of training plant seed portions to obtain training absorption data sets, the germination properties of the Plant seeds of the training plant seed portions are determined in subsequent germination property tests.

A method according to the invention is also preferred, wherein the germination properties determined in the subsequent germination property tests for the plant seeds of the training plant seed portions are corrected by a correction factor which takes into account the reduced germination properties as a result of incubation in the test composition, the correction factor being determined by correlation with that in germination property tests for Plant seeds from the same batch not used in the process are obtained.

The person skilled in the art readily understands that the training data set and the training on which the estimation module is based should sensibly correlate as largely as possible with the procedure of the method according to the invention. To put it bluntly, the person skilled in the art does not expect that an estimation module which was trained with a training data set which was created for plant seeds of type A1, incubation conditions B1 and wavelengths C1, can be usefully used in a method according to the invention which includes plant seeds of type A2 Incubation conditions B2 are treated and the wavelengths C2 are determined if A1, B1 and C1 are completely different from A2, B2 and C2. Consequently, preference is given to a method according to the invention, wherein the set of training data comprises a plurality of training absorption data sets which were obtained with method steps a) to d) of the method according to the invention for estimating the germination properties, essentially the same method parameters and/or devices being used.

• i) für Pflanzensamen erhalten wurden, die dem im Verfahren bereitgestellten Typ von Pflanzensamen entsprechen, und/oder
• ii) für Trainingspflanzensamenportionen erhalten wurden, die die gleiche Zahl von Pflanzensamen umfassen, wie die im Verfahren bereitgestellten Pflanzensamenportionen, und/oder
• iii) für Trainingstestsysteme erhalten wurden, in denen das gleiche Testvolumen an Testzusammensetzung eingesetzt wurde, wie in den im Verfahren erhaltenen Testsystemen, und/oder
• iv) für Trainingstestsysteme erhalten wurden, die unter den gleichen Bedingungen inkubiert wurden, wie die in dem Verfahren inkubierten Testsystemen, und/oder
• v) mit dem gleichen optischen Messverfahren, bevorzugt unter Verwendung der gleichen optischen Messvorrichtung, erhalten wurden, wie die im Verfahren erhaltenen Absorptionsdatensätze, und/oder
• vi) die gleichen Informationen über die Absorptionseigenschaften umfassen, wie die im Verfahren erhaltenen Absorptionsdatensätze, bevorzugt in der gleichen Datenstruktur. Wobei besonders bevorzugt 2 oder mehr, bevorzugt 3 oder mehr, besonders bevorzugt 4 oder mehr, ganz besonders bevorzugt 5 oder mehr, insbesondere sämtliche, dieser Maßgaben eingestellt werden.

In principle, in accordance with the understanding of the person skilled in the art, a method according to the invention is also particularly preferred, wherein the training absorption data sets of the set of training data:

• i) were obtained for plant seeds corresponding to the type of plant seeds provided in the process, and/or
• ii) training plant seed portions were obtained which comprise the same number of plant seeds as the plant seed portions provided in the method, and/or
• iii) were obtained for training test systems in which the same test volume of test composition was used as in the test systems obtained in the method, and / or
• iv) were obtained for training test systems that were incubated under the same conditions as the test systems incubated in the method, and / or
• v) were obtained with the same optical measurement method, preferably using the same optical measuring device, as the absorption data sets obtained in the method, and/or
• vi) include the same information about the absorption properties as the absorption data sets obtained in the method, preferably in the same data structure. Whereby particularly preferably 2 or more, preferably 3 or more, particularly preferably 4 or more, very particularly preferably 5 or more, in particular all of these requirements are set.

• i) Wasser,
• ii) einen zweistufigen Redox-Indikator,
• iii) einen fermentierenden Mikroorganismus, und
• iv) eine mit dem fermentierenden Mikroorganismus biokompatible Tensid-Verbindung, wobei die Tensid-Verbindung ausgewählt ist aus der Gruppe bestehend aus nicht-ionischen Tensiden. Diese Testzusammensetzung ist nach Einschätzung der Erfinder besonders vorteilhaft, weil sie eine ausgezeichnete Benetzung der Pflanzensamen mit der Testzusammensetzung gewährleistet, ohne dass auf mechanische Verfahren zu Steigerung der Benetzung zurückgegriffen werden müsste.

The invention also relates to a particularly preferred test composition for use in a method according to the invention, comprising:

• i) water,
• ii) a two-stage redox indicator,
• iii) a fermenting microorganism, and
• iv) a surfactant compound that is biocompatible with the fermenting microorganism, the surfactant compound being selected from the group consisting of non-ionic surfactants. According to the inventors, this test composition is particularly advantageous because it ensures excellent wetting of the plant seeds with the test composition without having to resort to mechanical methods to increase the wetting.

• A1) eine Ausgangsmischung, umfassend:
  • ii.b) einen zweistufigen Redox-Indikator,
  • iii.b) einen fermentierenden Mikroorganismus, und
  • iv.b) eine mit dem fermentierenden Mikroorganismus biokompatible Tensid-Verbindung, wobei die Tensid-Verbindung ausgewählt ist aus der Gruppe bestehend aus nicht-ionischen Tensiden, oder
• A2) in separaten Gebinden die Teilkomponenten zur Herstellung der Ausgangsmischung, sowie
• B) eine pflanzensamenspezifische Herstellungsanleitung, umfassend eine Herstellungsvorschrift zur Herstellung einer pflanzensamenspezifischen Testzusammensetzung durch Vermischen der Ausgangsmischung oder der Teilkomponenten der Ausgangsmischung mit einem wässrigen Lösungsmittel, insbesondere Wasser.

The invention also relates to a kit for producing a test composition according to the invention, comprising:

• A1) a starting mixture comprising:
  • ii.b) a two-stage redox indicator,
  • iii.b) a fermenting microorganism, and
  • iv.b) a surfactant compound that is biocompatible with the fermenting microorganism, the surfactant compound being selected from the group consisting of non-ionic surfactants, or
• A2) the sub-components for producing the starting mixture in separate containers, as well
• B) a plant seed-specific manufacturing instruction, comprising a manufacturing instruction for producing a plant seed-specific test composition by mixing the starting mixture or the partial components of the starting mixture with an aqueous solvent, in particular water.

According to the inventors' assessment, it is preferred to include additional components in the kit that may be useful to the end user when carrying out the method according to the invention.

For example, preference is given to a kit according to the invention, comprising a test plate with a plurality of test recesses for receiving test systems consisting of plant seed portions and test composition, the test plate preferably comprising 45 or more, preferably 90 or more, test recesses.

A kit according to the invention is also preferred, comprising a dosing and filling aid for filling a test plate with a large number of test recesses.

Also preferred is the kit according to the invention, comprising an optical measuring device, wherein the optical measuring device is set up to determine the optical absorption properties of the test composition for electromagnetic radiation of at least a first wavelength λ ₁ and a second wavelength λ ₂ , where λ ₁ and λ ₂ differ by 10 nm or more.

The invention further relates to a computer program product, comprising commands which, when the program is executed by an electronic data processing device, cause it to carry out method step e), preferably method steps d) and e), of the method according to the invention, the computer program product being based on machine learning Estimation module includes and is preferably stored on a portable storage unit, preferably on a USB-readable data carrier.

Also disclosed is an electronic data processing device for use in a method according to the invention for estimating the germination properties of the plant seeds in the plant seed portions, comprising a storage unit and a machine learning-based estimation module stored on the storage unit, wherein the electronic data processing device is set up in the inventive Method for giving the absorption data sets obtained for the plant seed portions as input into the estimation module and estimating the germination properties of the plant seeds in the plant seed portions with the estimation module, the estimation module being trained to estimate the germination properties of the plant seeds in the plant seed portions from the absorption data sets, the training being carried out by means of supervised Learning takes place with a set of training data, which includes a plurality of training absorption data sets of training plant seed portions of plant seeds with known germination properties.

• 1 eine schematische Flussdarstellung eines erfindungsgemäßen Verfahrens; und
• 2 ein Absorptionsspektrum von zwei RedOx-Zuständen des zweistufigen Redox-Indikators Resazurin.

The invention and preferred embodiments of the invention are explained and described in more detail below with reference to the accompanying figures. The figures show:

• 1 a schematic flow representation of a method according to the invention; and
• 2 an absorption spectrum of two RedOx states of the two-stage redox indicator resazurin.

1 shows a schematic representation of the process steps of the method according to the invention for estimating the germination properties of plant seeds in a preferred embodiment. The germination properties, germination ability and driving force are estimated according to Ista criteria.

In method step a) 100 of the exemplary method according to the invention, 80 separate plant seed portions are provided, each of which consists of exactly one Brassica napus plant seed, that is, a rapeseed. Here, the plant seed portions are arranged individually in test recesses in a so-called multiwell plate.

In process step b) 102, a test composition is produced which, based on the mass of the test composition, contains 99.9705% of distilled water, 0.0005% resazurin and 0.004% Saccharomyces cerevisiae as well as 0.025% of a surfactant compound that is biocompatible with the microorganism, which is below Trade name BreakThru SD260 is available from Evonik Operations GmbH.

The fermenting microorganism is presented in a freeze-dried state with the resazurin and the surfactant compound as a powder, which is mixed with cold water (approx. 6 °C) to produce the test composition according to plant seed-specific instructions. The test composition is produced immediately before method step c) 104, so that there is only a small time delay between the production of the test composition and contact with the plant seed portions. This advantageously makes it possible to largely avoid intermediate heating of the test composition and the occurrence of signs of aging. The surfactant compound used achieves rapid, complete wetting of the plant seeds with the test composition in method step c) 104 of the method according to the invention.

In process step c) 104, the plant seed portions are each contacted with a test volume of 150 µl of test composition in order to obtain 80 separate test systems. The multiwell plate with the test systems is then incubated with the exclusion of light for a period of 4 hours and at a temperature of around 21 °C. Following the incubation, 100 µL of test composition is added taken from each test system and transferred to a new multiwell plate for subsequent measurement.

In addition to the 80 test systems, in method step c) 104 a total of 4 blind systems, which contain exclusively 150 µl of distilled water, as well as a total of 4 control systems, which contain 0.3125 mmol of a first carbohydrate compound (namely sucrose) in the test composition, 4 control systems, which contain 0.078125 mmol of a second carbohydrate compound (namely sucrose) in the test composition, and 4 reference systems, which contain exclusively 150 µl test composition, are generated and incubated with the test systems on the same multiwell plate.

In process step d) 106, the test compositions of the incubated test systems as well as the blank, control and reference systems are finally measured with an Absorbance 96 photometer from Byonoy GmbH in a transmission measurement process to determine the optical absorption properties (OD values) of the respective test compositions for electromagnetic Radiation is measured at a first wavelength λ ₁ = 600 nm and a second wavelength λ ₂ = 570 nm. This results in a number of absorption data sets corresponding to the number of test systems. The absorption data sets contain corresponding information about the absorption properties of the respective test compositions at 600 and 570 nm, with the values derived from the corresponding absorption values being further processed, which were corrected taking into account the blind measurements or normalized the control measurements.

In method step e) 108, the absorption data sets are analyzed using a computer to estimate the germination properties of the plant seeds in the respective plant seed portions. For this purpose, software with an estimation module is used, which is based on a machine learning algorithm, which was trained for this purpose by means of supervised learning with a set of training data from training absorption data sets of training plant seed portions of rapeseed with known germination properties, as disclosed above.

In method step f) 110, an average germination property forecast in the form of a box plot is then calculated by averaging the germination properties estimated for the plant seed portions. In method step g) 112, this estimated average germination property prognosis is output via an electronic one.

Table 3 summarizes an exemplary set of training data that was detected using the method steps a) 100 to d) 108 described above for four sets of 80 plant seeds each. Table 3 includes the normalized and corrected absorptions at 570 nm (A1) and 600 nm (A2) for the plant seeds (No.). In addition, for the purpose of supervised learning, the corresponding result of the sowing test is entered, i.e. whether the plant seeds germinated physiologically (P) and have formed a normal seedling (N) (1 = Yes / 0 = No).

For example, for different plant seeds or different batches of seeds, the inventors compared the accuracy of the estimate of the method according to the invention (ACC600/570) with the accuracy of the estimate obtained when evaluating only one wavelength (ACC570 and ACC600). For this purpose, training takes place with 80% of the total data available. The remaining 20% are presented to the trained estimation module and compared with the actual germination success (P and N each, also called “ground truth”) (predictions >= 0.5 are rated as 1, predictions <0.5 are rated as 0 ; The proportion of correct predictions in percent is the “Accuracy”). For Triticum aestivum, the influence of fungicide treatment was also estimated. For this purpose, samples from the same batch that were untreated and treated with fungicide (trade name: Vibrance Trio; Syngenta company) as well as a mixture (“partially treated with fungicide”, ratio: 1:1) were subjected to the process. The estimation module used was, on the one hand, separated on the treated and untreated seeds and, on the other hand, trained on the entire treated/untreated seed.

The results for germination and driving force are summarized in Tables 1 and 2. Table 1 - “Accuracy” values for estimating germination Model Acc600/570 Acc600 Acc570 Beta vulgaris 0.779 0.723 0.718 Hordeum vulgare 0.767 0.689 0.697 Triticum aestivum (partially treated with fungicide) 0.827 0.743 0.739 Triticum aestivum (fungicide treated) 0.800 0.778 0.778 Triticum aestivum (partially treated with fungicide) 0.820 0.685 0.693 Brassica napus 0.874 0.865 0.863 Brassica napus 0.875 0.831 0.828 Triticum aestivum (untreated) 0.810 0.688 0.730 Brassica oleracea 0.902 0.867 0.891 Triticum aestivum (untreated) 0.885 0.661 0.730 Triticum aestivum (fungicide treated) 0.878 0.722 0.662 Brassica oleracea 0.896 0.857 0.836 Beta vulgaris 0.909 0.630 0.635 Table 2 - “Accuracy” values of the driving force estimation Model Acc600/570 Acc600 Acc570 Beta vulgaris 0.688 0.524 0.576 Hordeum vulgare 0.781 0.714 0.759 Triticum aestivum (partially treated with fungicide) 0.788 0.719 0.730 Triticum aestivum 0.800 0.769 0.784 (Fungicide treated) Tritcum aestivum (partially treated with fungicide) 0.812 0.695 0.699 Brassica napus 0.827 0.810 0.804 Brassica napus 0.827 0.794 0.812 Triticum aestivum (untreated) 0.835 0.668 0.747 Brassica oleracea 0.864 0.823 0.853 Triticum aestivum (untreated) 0.878 0.661 0.714 Triticum aestivum (fungicide treated) 0.887 0.709 0.691 Brassica oleracea 0.891 0.836 0.841 Beta vulgaris 0.932 0.659 0.788

The estimation according to the method according to the invention consistently shows a significantly improved estimation quality and provides the best estimate in all cases. It should be noted here that the comparison values ACC600 and ACC570 compared to the method known from the prior art were also evaluated using an estimation module based on machine learning, so that the comparison values shown here compared to the comparatively simple regression methods known from the prior art already deliver significantly improved estimation qualities.

In 2 To illustrate the effect of a change in the redox indicator, absorption spectra of the two-stage redox indicator resazurin are shown graphically in different redox states A and B, with the absorption on the Y-axis and the wavelength in nanometers on the X-axis. The absorption spectrum A was measured on resazurin and has an absorption maximum at 600 nm. The absorption spectrum B was measured on resorufin, i.e. reduced resazurin, and has an absorption maximum at 570 nm. The reduction of resazurin to resofurin correspondingly causes a shift in the absorption maximum by approximately Δ = 30 nm. The determination of the absorption properties at the two absorption maxima is particularly efficient in the method according to the invention since, as shown 2 It can be seen that at these wavelengths there are particularly pronounced differences between the absorptions of the two components.

Table 3 - Example training data set

Reference symbol list

100

Process step a)

102

Process step b)

104

Process step c)

106

Process step d)

108

Process step e)

110

Process step f)

112

Process step g)

Claims (10)

Method for estimating the germination properties of plant seeds, comprising the method steps: a) providing a plurality of separate plant seed portions each comprising at least one plant seed, b) producing or providing a test composition comprising: i) water, ii) a two-stage redox indicator, and iii ) a fermenting microorganism, c) contacting the plant seed portions with a test volume of test composition to obtain a large number of separate test systems and incubating the test systems, d) measuring the test compositions of the incubated test systems using an optical measuring method to determine the optical absorption properties of the respective test compositions for electromagnetic Radiation of at least a first wavelength λ ₁ and a second wavelength λ ₂ to obtain a plurality of absorption data sets assigned to the respective test systems, the absorption data sets comprising information about the absorption properties of the respective test compositions at the first wavelength λ ₁ and the second wavelength λ ₂ , wherein λ ₁ and λ ₂ differ by 10 nm or more, e) evaluating the absorption data sets assigned to the test systems for estimating the germination properties of the plant seeds in the respective plant seed portions with an electronic data processing device, the electronic data processing device comprising a storage unit, an estimation module based on machine learning being stored on the storage unit, the electronic data processing device being set up for this purpose is to enter the absorption data sets obtained for the plant seed portions as input into the estimation module and to estimate the germination properties of the plant seeds in the plant seed portions with the estimation module, the estimation module being trained to estimate the germination properties of the plant seeds in the plant seed portions from the absorption data sets, the training by means of supervised learning with a set of training data, which includes a large number of training absorption data sets of training plant seed portions of plant seeds with known germination properties. Procedure according to Claim 1 , where the two-stage redox indicator is resazurin. Procedure according to one of the Claims 1 or 2 , where the fermenting microorganism is selected from the group consisting of unicellular fungi. Procedure according to one of the Claims 1 until 3 , wherein the test composition additionally comprises one or more surfactant compounds biocompatible with the fermenting microorganism. Procedure according to one of the Claims 1 until 4 , whereby the surfactant compound is selected from the group consisting of surfactants that are compatible with the fermenting microorganism. Procedure according to one of the Claims 1 until 5 , where the first wavelength λ ₁ and the second wavelength λ ₂ differ by 15 nm or more. Procedure according to one of the Claims 1 until 6 , where the first wavelength λ ₁ is in the range from 585 to 630 nm, and/or where the second wavelength λ ₂ is in the range from 540 to 585 nm. Test composition for use in a method according to one of the Claims 1 until 7 , comprising: i) water, ii) a two-stage redox indicator, iii) a fermenting microorganism, and iv) a surfactant compound biocompatible with the fermenting microorganism, the surfactant compound being selected from the group consisting of non-ionic surfactants . Kit for preparing a test composition Claim 8 , comprising: A1) a starting mixture comprising: ii.b) a two-stage redox indicator, iii.b) a fermenting microorganism, and iv.b) a surfactant compound biocompatible with the fermenting microorganism, the surfactant compound being selected from the group consisting of non-ionic surfactants, or A2) in separate containers the sub-components for producing the starting mixture, and B) a plant seed-specific manufacturing instruction, comprising a manufacturing instruction for producing a plant seed-specific test composition by mixing the starting mixture with an aqueous solvent. Computer program product, comprising commands which, when the program is executed by an electronic data processing device, cause it to carry out method step e), preferably method steps d) and e), of the method according to one of the Claims 1 until 8th to execute, wherein the computer program product comprises the machine learning-based estimation module.

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